Capillary electrophoresis based affinity assay for screening immunomodulating drug candidates

Abnormal immune response causes a large number of severe health disorders which must be closely controlled to sustain a normal immune system. Immunomodulators are substances that are able to control an abnormal immune response. The search for novel and enhanced immunomodulators is, therefore, one of the mainstream directions in modern drug discovery. Binding of the drug candidate to its therapeutic target is the initial and indispensable step in the cascade of reactions that finally cause a pharmacological effect. Many successful and widely used techniques in drug discovery are thus based on measuring target-ligand binding. In the following work, capillary electrophoresis-based competitive affinity assay was developed for preliminary screening of novel immunomodulators. The screening approach involved the use of CD4 glycoprotein as a therapeutic target and fluorescently labelled monoclonal CD4 antibody as an affinity probe with which immunomodulators competed for binding the target. The developed affinity assay was successfully validated with CD4 ligands such as: non-labelled analog of CD4 antibody, an envelope protein of HIV-1 (human recombinant gp 120), and small molecule of green tea extract (Epigallocatechin gallate). The screening of 14 selected immunomodulating peptides resulted in no apparent affinity for CD4 glycoprotein. To technologically advance the developed affinity assay and reduce reagent consumption, in-capillary mixing by transverse Diffusion of Laminar Flow Profiles (TDLFP) was used for the screening of CD4 ligands. Finally, the use of CD4 aptamers instead of antibodies for the screening of immunomodulators in the future was suggested. An approach for screening aptamer clones which combines asymmetric PCR amplification and TDLFP-Kinetic Capillary Electrophoresis (KCE) based affinity analysis was developed (Yunusov, 2009). For the first time, it was shown that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. It was also shown that mathematical modeling of TDLFP based mixing allows for accurate affinity ranking of aptamer clones based on the calculated Kd values.Keywords. Affinity assay Antibody Aptamers Capillary electrophoresis CD4 glycoprotein Immunomodulators Kinetic Capillary Electrophoresis (KCE) Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixture (NECEEM) Short peptides Transverse Diffusion of Laminar Flow Profiles (TDLFP).